The present invention relates generally to the field of administration of superoxide dismutase (SOD) to reduce ischemic injury or injury following sepsis or inflammation. In particular, this invention relates to a method and compositions for reducing toxic side effects caused by the reaction of SOD and peroxynitrite. Specifically, an effective amount of SOD, modified by substituting amino acid residues close to the active site of the SOD with amino acid residues, such as tyrosine, methionine, or cysteine residues, that can trap the toxic side products of the reaction of SOD with peroxynitrite, such as nitronium ion, is administered to an animal. This invention has particular relevance in the treatment of stroke and head trauma, myocardial ischemia, sepsis, inflammation, adult respiratory distress syndrome, and bronchiopulmonary dysplasia.
Peroxynitrite anion (ONOO.sup.31) is a potent oxidant. Peroxynitrite is formed by the reaction of superoxide (O.sub.2.sup.31) and nitric oxide in tissues subjected to ischemic, inflammatory or septic conditions. Nitric oxide is present in such tissues. For example, in ischemic injury, ischemia allows calcium entry into endothelial cytoplasm due to failure of ionic pumps and opening of ion channels. Endothelium and neurons produce nitric oxide by an oxygen (O.sub.2) dependent calmodulin activated nitric oxide synthetase which oxidizes arginine in the presence of NADPH (Palmer et al., Nature (London) 333:664-666 (1988); Knowles et al., Proc. Natl. Acad. Sci. (USA) 86:5159-62 (1989); Marletta et al., Biochem., 27:8706-8711 (1988)). Reperfusion allows rapid nitric oxide synthesis by providing O.sub.2 to the enzyme and other substrates already present as a result of ischemia.
Superoxide is also present in injured tissue. For example, ischemia induces intracellular O.sub.2 production by xanthine oxidase, mitochondria and other sources. The O.sub.2 can escape into the extracellular millieu through anion channels (Lynch et al., J. Biol. Chem., 253: 4697-4699 (1978)). Extracellular O.sub.2.sup.- and nitric oxide are also produced in the vascular lumen by activated neutrophils and macrophages, and by circulating xanthine oxidase released from liver (Yokoyama et al., Amer. J. Physiol., 258:G564-G570 (1990); Moncada et al., Biochem. Pharmacol., 38:1709-1715 (1989)). The superoxide radical is also an important mediator of both the inflammatory response of neutrophils and of the damage that occurs during reperfusion of anoxic tissue after organ transplantation or when a blood clot is removed. (Petrone et al., Proc. Natl. Acad. Sci (USA), 77:1159-163 (1980)).
NO reacts rapidly with O.sub.2.sup.- both intracellularly and in the vascular lumen to form peroxynitrite (Blough et al., Inorg. Chem., 24:3504-3505 (1985); Beckman et al., Proc. Natl. Acad. Sci. (USA), 87:1620-1624 (1990)). The rate of peroxynitrite formation depends upon O.sub.2.sup.- and nitric oxide concentrations. Peroxynitrite can be toxic by at least three mechanisms: hydrogen ion-catalyzed homolytic cleavage to form hydroxyl radical (.cndot.OH) and nitrogen dioxide (NO.sub.2), direct reaction with sulfhydryl groups, and reactions with SOD and transition metals to form hydroxyl ion (.sup.- OH) and nitronium ion (NO.sub.2.sup.+) a potent nitrating agent (Beckman et al., Nature (London) 345:27-28 (1990). Thus, peroxynitrite is a reactive species which can produce other highly reactive species such as .cndot.OH, NO.sub.2, and NO.sub.2.sup.+.
Superoxide dismutases consist of several distinct families of metal-containing enzymes that catalyze the dismutation of the oxygen radical superoxide by the following two step reaction: EQU O.sub.2.sup.- +Me-SOD====.fwdarw.O.sub.2 +Me.sup.-1 -SOD EQU 2H.sup.+ +O.sub.2.sup.- +Me.sup.-1 -SOD====.fwdarw.H.sub.2 O.sub.2 +Me-SOD
where Me refers to the metal bound in the active site. This metal undergoes repeated cycles of oxidation and reduction in the reactions given above. Many compounds can either reduce or oxidize superoxide, but the distinguishing feature of a superoxide dismutase is the catalysis of both reactions given above. A series of positively charged amino acids positioned near the active site of the SODs generates an electrostatic gradient which attracts the negatively charged O.sub.2.sup.- into the active site.
Three families of SODs are distinguished by the metal in the active site: the copper+zinc (Cu,Zn) SOD family, the manganese (Mn) SOD family and the iron (Fe) SOD family. The vast majority of therapeutic studies have been performed using Cu,Zn SODs, which naturally occur in the cytoplasm of eukaryotic cells as a dimer of two identical 16 Kd peptides. There is also a distinct Cu,Zn SOD found in plasma, which is a tetramer. Another form of SOD contains manganese (Mn) and is found in mitochondria. This protein has an amino acid sequence distinct from the Cu,Zn SOD but is similar to the sequence for Mn and Fe SODs found in bacteria.
SOD's have been commonly utilized to prevent or reduce oxidation injury in the treatment of stroke and head trauma, myocardial ischemia, abdominal vascular occlusion, cystitis, and a variety of inflammatory conditions (Greenwald, Free Radical Biol. and Med., 8:201-209 (1990); McCord, New Eng. J. Med., 312:159-183 (1985); U.S. Pat. No. 4,695,456; U.S. Pat. No. 4,656,034).
However, there have been several disappointing results in humans treated with native human Cu,Zn SOD. One explanation for these results may be that the native enzyme has a circulatory half life of only minutes because of rapid clearance by the kidneys (Petkau et al., Res. Commun. Chem. Pathol. Pharmacol., 15:641-657 (1976)).
To address this problem, several longer-lived derivatives of SODs have been developed. Human Mn SOD has been cloned and human trials have been reported by Nimrod et al., in Medical Biochem. and Chem. Aspects of Free Radicals, N.Y.: Elsevier Science Pub., 743-746 (1989). Genetic engineering has produced a long-lived modification of human cytoplasmic Cu,Zn SOD (Hallewell et al., J. Biol. Chem., 264:5260-5268 (1989)). Other modifications, such as polyethylene glycol conjugates of both human and bovine Cu,Zn SOD have longer half lives and are less immunogenic than unmodified forms (Pyatak et al., Res. Commun. Chem. Pathol. Pharmacol., 29:113-127 (1980); Saifer et al., Proc. Fifth Internatl. Conf. on Suoeroxide and Suoeroxide Dismutase (Jerusalem) (1989)).
Another problem with in vivo SOD therapy has recently been reported in which treatment with higher SOD dosage levels to reduce ischemic injury resulted in an increased infarct size (Omar et al., Circulation 80:SII-294 (1989); Werns et al., Tr. Pharmacol. Sci., 1:161-166 (1988)). Further, the use of SOD in tissues containing ONOO.sup.- can lead to the production of other destructive species, such as NO.sub.2.sup.+. Thus, there exists a need for an improved therapy to treat ischemic, inflammatory or septic conditions.